Introduction

The aquaculture and fisheries industry is one of the most important sources of animal proteins for human consumption (Maulu et al. 2021; Eissa et al. 2022a). Oreochromis niloticus is one of the highest cultivated fish in Egypt and globally (Eissa et al. 2022b; Khalil et al. 2022; Eissa et al. 2022c). Tilapia production is one of the world’s fastest-expanding aquaculture sector (Khalil et al. 2021; Hendam et al. 2023). In 2017, Egypt produced 15.4% of the world’s tilapia production, totalling 6,510,700 tonnes (El Basuini et al., 2021). Due to the growing demand for animal protein, intense agricultural activities are required to obtain the desired level of production (Mansour et al. 2021). Aquaculture is subject to exotic, endemic pathogens (mainly parasites and bacteria) (Eissa et al. 2021; Eissa et al. 2023a; Eissa et al. 2023b), as well as climatic and environmental disruptions (Eissa et al. 2022c; Abu-Elala et al. 2016). As a result, developing resistant tilapia that can fight infectious illnesses and attain better growth rates is of significant importance (Kord et al. 2021a). The concept of functional foods is currently being employed in the food industry for the production of animals since diets must meet nutritional requirements as well as support the health of farmed animals such as fish (El Basuini et al. 2021; Eissa et al. 2022d). A nutrient-rich aqua feed should comprise minerals and vitamins, as well as protein, lipids and carbohydrates (Van Doan et al. 2020).

Trace minerals are also connected to several physiological, metabolic and functional features of an organism’s body (Khan et al. 2017). Immuno-nutrition covers fish additives and/or essential trace elements to boost their growth, antioxidant defences and immunological responses (Eissa et al. 2023a). Selenium is a trace mineral that has recently gained much interest in animal nutrition and health (Khalil et al. 2019). Selenium is a necessary component of the non-enzymatic antioxidant system, as well as glutathione peroxidase, which shields the cell membrane from oxidative damage (Talas et al. 2008).

Nanotechnology provides unique ways to raise the safety, absorption and bioavailability of chemicals in aquaculture due to their small size and large surface area (Khalil et al. 2023). Selenium nanoparticles (Se-NPs) were reported to promote an efficient antioxidant defence system, and growth performance of common carp (Cyprinus carpio) compared to selenomethionine or sodium selenite as it increases absorption and bioavailability (Saffari et al. 2018). In fish, Se-NPs boost biochemical markers (growth hormone levels in serum, total protein content in tissue and glutathione (GSH) peroxidase activity in the muscle and liver as well as haematological parameters as RBC count, haemoglobin concentration, haematocrit values and lysozyme activity (Khan et al. 2016). Se-NPs increased skin mucus, serum total proteins, serum and mucus peroxidase, mucus lysozyme activity, tolerance against low salinity and the alternative complement pathway in red sea bream (Dawood et al. 2019).

The previous research confirmed a link between fish mycoses and Aspergillus spp. (Saleemi et al. 2020). Aspergillosis was thought to be caused by Aspergillus terreus and Aspergillus flavus, and it was discovered in Labeo calbasu. Fish mucus is where Aspergillus spores are principally located. In the mucus of silver carp (Hypophthalmichthys molitrix), spores of Aspergillus niger and Aspergillus flavus were discovered (Balasubramanian et al. 2012). In the current decade, aquatic feeds should be extensively researched to serve as a functional nutritional supplement to enhance growth, immune responses and disease resistance of aquatic organisms (Abdel-Latif et al. 2022). Many researchers determined the effects of selenium nanoparticles on the growth performance and nutritional quality in Nile tilapia, Oreochromis niloticus, focusing on flesh quality and lipid fatty acid stability (Dawood et al. 2019; Saleemi et al. 2020; Balasubramanian et al. 2012; Abdel-Latif et al. 2022; Kokila et al. 2017; Dawit Moges et al. 2022).

Thus, the present work was aimed to investigate the impacts of Nano-selenium particles on performance, digestive enzyme activity, antioxidant status and immune resistance of Nile tilapia Oreochromis niloticus when challenged with Aspergillus flavus infection

Material and methods

Synthesis of selenium nanoparticles

Se-NPs were made following Dawood et al. (Dawood et al. 2019) with some adjustments. The stock solutions of sodium selenite (100 mM) and ascorbic acid (50 mM) were formed. The reacted sodium selenite to ascorbic acid ratio was 1:3 from the primary stock solution. The ascorbic acid solution was added dropwise to the sodium selenite solution for 30 min at laboratory temperature while being magnetically stirred. The combinations were then allowed to react until a light red colour was observed instead of a colourless one. The prepared Se-NP was coated with Dextrin at a level of 5% which was applied after the rise of colour using a magnetic stirrer at laboratory temperature using a single-layer coating approach. The produced nanoparticles were diluted using the dextrin solution instead of utilizing water. Finally, the nanoparticles were dried using a freeze-dryer. The formed nanoparticles were characterized using the transmission electron microscope (TEM) presented in Fig. 1. The TEM Se-NPs were prepared for TEM analysis by placing a drop of the nanoparticle suspension on carbon-coated copper grids according to (Kokila et al. 2017).

Fig. 1
figure 1

A Characterization of Se-NP under transmission electron microscopy; B size distribution of Se-NP report by intensity; C size distribution of Se-NP report by number; D size statistics report by number

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Fish rearing and experimental diets

Table 1 represents the chemical composition of the control diet. Four identical diets were formulated except for different levels of Se-NPs, a control diet with 30% crude protein (CP). Se-NPs were added to the control diet at amounts of 0.0 (control), 0.5 (T1), 1.0 (T2) and 1.5 (T3) mg/kg diet have been done according to Dawit Moges et al. (Dawit Moges et al. 2022). Se-NP was suspended in 100 mL distilled water, uniformly sprayed onto diet ingredients, well-mixed for 30 min and pelleted (1–2-mL diameter). Diets were prepared and stored in plastic bags at 4 °C until needed. Nile tilapia fingerlings (4.5 ± 0.5 g) were housed in the lab for 2 weeks to acclimate and adjust to the environment. During that time, they were given the control diet of thirty percent (30%) CP up to apparent satiation three times a day. Fish were randomly distributed in 12 tanks 100 L at a density of 20 fish per tank after acclimation to represent four treatments in triplicates. Compressed air was supplied to the fish tanks using air stones and air pumps. For 60 days, fish were fed the experimental diets three times a day at 9:00 h with 4-h intervals until they appeared satiated. Half of the water in each tank was withdrawn daily, along with faeces, and changed with new well-aerated water from a stock during the adaptation. Using fluorescent light tubes, the light was kept at a 12-h:12-h light and dark cycle throughout the experiment.

Table 1 Ingredients and proximate chemical composition of the control diet (g/kg dry matter basis)
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Water quality

Water samples were taken for the physicochemical test at 15-day intervals. Dissolved oxygen, water temperature, un-ionised ammonia, salinity, NO2, NO3 and pH were recorded on sites using Hanna HI-9147 automatic probe according to APHA (APHA, 2012). Nile tilapia’s fingerlings water quality parameters were rearing according to Kord et al. (Kord et al. 2021b).

Growth indices and fish survival

All fish in the experimental groups were weighed at the end of feeding period to estimate growth variables, including total length (TL) and standard length (SL). Weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) were computed using the formulas.

$$\textrm{WG}\ \left(\textrm{g}\right)={\textrm{W}}_{60\ \textrm{DAY}}-{\textrm{W}}_{0\ \textrm{DAY}}$$
$$\textrm{Weight}\ \textrm{gain}\%=100\times \left[\left({\textrm{W}}_{60\ \textrm{DAY}}-{\textrm{W}}_{0\ \textrm{DAY}}\right)/{\textrm{W}}_{0\ \textrm{DAY}}\right]$$
$$\textrm{SGR}\ \left(\%/\textrm{day}\right)=100\times \left[\left(\textrm{Ln}\ {\textrm{W}}_{60\ \textrm{DAY}}-\textrm{Ln}\ {\textrm{W}}_{0\ \textrm{DAY}}\right)/60\right]$$

where W60 DAY is the final weight (g), W0 DAY is the opening weight (g) and 60 is the trial period (day)

$$\textrm{FCR}=\textrm{Feed}\ \textrm{intake}/\textrm{WG}$$
$$\textrm{Survival}\ \left(\%\right)=100\times \left(\textrm{Fish}\ {\textrm{number}}_{60\ \textrm{DAY}}/\textrm{Fish}\ {\textrm{number}}_{0\ \textrm{DAY}}\right).$$

Three fish were randomly chosen from each tank after the final weighing to ascertain the proximate composition using standard methods. Crude protein was estimated by nitrogen analysis (N*6.25) using the Kjeldahl technique. Crude lipid was assessed by the Soxhlet petroleum ether extraction method. Moisture was determined by drying samples at 105 °C to a constant weight. Ash was determined by burning at 550 °C for 24 h according to AOAC (AOAC 2016).

Blood sampling

Feeding was stopped 24 h before blood samples were drawn. Sodium bicarbonate-buffered tricaine methanesulfonate (MS222; Sigma-Aldrich, USA) (30 mg/L) was used to anaesthetize five fish per replicate. After that, blood samples were taken from the caudal fish vessels and divided into two parts. The first one with sodium citrate as an anticoagulant was analysed the same day to determine haematological parameters. The second part was allowed to clot at room temperature in clean, dry centrifuge tubes before being centrifuged at 5000 × g for 15 min at room temperature to obtain sera.

Haemato-biochemical examination

A haemocytometer was used to count WBCs and RBCs, according to Brown (Brown 1980). The cyanmethaemoglobin method, developed by Jain (Jain 1986), was used to calculate haemoglobin (Hb) and haematocrit (Ht) levels, respectively. An Olympus oil-immersion light microscope with ×1000 magnification was also used to count lymphocytes, neutrophils and monocytes. Trinder’s methods assessed blood glucose levels (Trinder 1969). The method of Fossati (Fossati & Prencipe 1982) was used to determine serum cholesterol and triglycerides, respectively. Henry’s methods for determining total protein (TP) and albumin (ALB) in serum were used.

Digestive enzyme activities

Diagnostic reagent kits from Cusabio Biotech Co. Ltd., Wuhan, Hubei, China, were used to assess the activities of digestive enzymes in fish serum according to the producer’s guidelines. Bernfeld (Bernfeld 1955), Shihabi (Shihabi & Bishop 1971) and updated by Zaki et al. (Zaki et al. 2023), as a described method for determining amylase, lipase and protease activity, respectively.

Antioxidants and immunity biomarkers

Using diagnostic reagent kits, the antioxidant biomarkers were determined (Bio-diagnostic Co., Giza, Egypt). The method of Ohkawa et al. (Ohkawa et al. 1979) was applied to evaluate the amount of malondialdehyde (MDA) in the samples. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined using McCord (McCord & Fridovich 1969), Aebi (Aebi 1984) and Paglia, Valentine (Paglia & Valentine 1967) techniques, respectively. The turbidimetric approach was used to assess serum lysozyme (LYZ) in phosphate buffer (pH = 6.2) with Micrococcus luteus as the target (Ellis 1990). IgM was quantified after polyethylene glycol precipitation of IgM, and removal of the initial and final total protein was done.

Challenge test with Aspergillus flavus

Aspergillus flavus strain

Aspergillus flavus strain was provided from the Microbiological Unit of Fish Diseases Department, Animal Health Institute, ARC Dokki, Giza province. The definition was confirmed in the mycology lab., Botany Department, Faculty of Sciences, Arish University.

Preparation of fungal spores suspension

Aspergillus flavus fungal strains cultivated on Sabourauds dextrose agar (SDA, Difco). One hundred (100) UI/mL penicillin and 100 ug/mL streptomycin were added to the medium. The conidia were gathered by adding 20-mL sterile distilled water to each culture plate and collecting the solution in sterile 30-mL autoclave tubes after a 7-day incubation period at 25 °C. To verify that the filtrate contains fungal conidia, the suspensions were filtered through two layers of sterile medical gauze. Thereafter, the concentrations were adjusted to 4 × 103 conidia/mL sterile distilled water using a red blood cell counting chamber (Willoughby & Roberts 1994). Ten fish were taken from each group and injected in I/P with 0.2 mL of concentrations 4 × 103 of Aspergillus flavus conidial suspension and monitored for 15 days. During this period, clinical signs, post-mortem lesions and mortality were recorded.

Histological analysis

At the end of the experiment, all fish fasted for 24 h. Subsequently, terminal anaesthesia (MS222; Sigma-Aldrich, USA; 30 mg/L) was used for five fish taken from each tank (n = 15 fish per treatment). Thereafter, collected tissue specimens were taken from the liver, intestine, gills and skin. The collected samples were fixed 10% buffered formalin for 24 h, dehydrated in the ascending grade of ethyl alcohol (50–99%), cleared in xylene, embedded in Parablast wax, cutting by a rotatory microtome (Leica, UK) at 5 μm in thickness and then used the haematoxylin and eosin (H&E) for staining the prepared slides and examined ten fields for five slides from each group under the light microscope (Leica, UK) (Eissa et al. 2023b).

Statistical analysis

The data were computed and presented as means ± standard error using SPSS version 23 by one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparisons to compare the means between individual treatments at the P level of < 0.05 according to Dytham (Dytham 2011).

Results

Water quality parameters

All estimated water quality parameters were within the normal range. There were no differences in temperature, salinity, pH, DO, NH4, NO2 and NO3 in different treatments (Table 2).

Table 2 Effect of nano-selenium levels on water quality parameters of Nile tilapia, O. niloticus after 60 days
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Growth indices and fish survival

The growth performance and feed utilization in terms of final weight (g), wet gain (g), wet gain (%), SGR (%/day), FCR and final length (without tail and with tail) of Tilapia fingerlings recorded were significantly higher (P ≤ 0.05) in T2 followed by T1 and T3 diets than in the control group (Table 3). The survival rate was not significant (P ≥ 0.05) in different treatments with selenium nanoparticles.

Table 3 Effect of nano-selenium levels on growth performance parameters of Nile tilapia, O. niloticus after 60 days
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Proximate composition of the fish body

The proximate composition of tilapia fingerlings displayed a significant increase in moisture content (Table 4, P ≤ 0.05). Crude protein was recorded considerably higher values (P ≤ 0.05) in T3, followed by control, then T1 and finally T2. In terms of the lipid, there was a significant elevation in T2 followed by T1, T3 and control. Ash content was significantly higher (P ≤ 0.05) in the control than in other treatments. However, there was no major difference between T2 and T3 (P ≥ 0.05). Selenium residues in tissues of O. niloticus increased significantly in selenium treatments than in the control group and the highest value was recorded in T3 (Table 4).

Table 4 Effect of nano-selenium levels on chemical body composition parameters of Nile tilapia, O. niloticus after 60 days
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Haemato-biochemical parameters

No significant difference in haemoglobin, HCT, MCH, MCHC, MCV, WBCs, RBCs, total protein, albumin, ALT, AST, ALP, creatinine, glucose, uric acid, cholesterol and triglyceride between different groups. Lymphocytes and neutrophils were significantly raised (P ≤ 0.05) in the control more than in other treatments (Table 5). In terms of the monocytes, there were significantly higher in T3 than in the control group but there were no differences in T1, T2 and T3 as shown in Table 5. Globulin increased significantly in fish fed diets supplemented with NSP versus the control group in Table 6.

Table 5 Effect of nano-selenium levels on haematological parameters of Nile tilapia, O. niloticus after 60 days
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Table 6 Effect of nano-selenium levels on haemato-biochemical parameters of Nile tilapia, O. niloticus after 60 days
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Digestive enzyme activities

There were no substantial alterations in amylase and protease among different groups. Lipase was increased significantly (P ≤ 0.05) in diets supplemented with selenium compared to the control group, while there were no differences among selenium groups (Table 7).

Table 7 Effect of nano-selenium levels on digestive enzyme activities parameters of Nile tilapia, O. niloticus after 60 days
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Antioxidants and immunity biomarkers

The values of the antioxidants and immunity biomarkers are presented in Table 8. MDA, SOD, CAT, lysozyme and IgM were significantly lower in control than other treatments. There was no difference in GPx measurements between different groups.

Table 8 Effect of nano-selenium levels on antioxidant and immunity biomarkers parameters of Nile tilapia, O. niloticus after 60 days
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Challenge test with Aspergillus flavus

The clinical signs of infection in fish were abnormal swimming behaviour, high mortality rate, dark skin, slight flatulence and exophthalmos with corneal opacity (Fig. 2A). Post-mortem pathological changes include some haemorrhages on the surface of the body in the form of creamy haemorrhage tending to haemorrhagic fluid in the abdominal cavity, congestion and ulceration of the gills, congestion and haemorrhage on the surfaces of the internal organs along with black necrotic foci on the liver, and distention of the gallbladder (Fig. 2B). The recorded mortality was 70%, 60%, 40.33% and 40.67% in control, T1, T2 and T3 groups, respectively (Table 9). Fungal reisolation from all infected fish gills, livers, spleen, kidneys and the muscle of artificially infected fish showed increased resistance.

Fig. 2
figure 2

A A clinical signs: Nile tilapia (O. niloticus) showing skin darkening, slight abdominal inflation, exophthalmia and corneal opacity. B P. M. lesions: Nile tilapia (O. niloticus) showing erosion of gills, congestion and haemorrhages on the surfaces of internal organs with black necrotic foci on the liver

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Table 9 Effect of nano-selenium levels on the mortality percentage of Nile tilapia, O. niloticus post-challenge with A. flavus infection
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Histological examination

The sections of hepatopancreatic tissues for Nile tilapia are described in Fig. 3. The control group (T0) showed marked degeneration of hepatocytes. The pancreatic islands showed mild congestion, degeneration and dilation (Fig. 3A). T1 is explained the moderate congestion, degeneration, moderate dilation in hepatic sinusoids and inflammatory cells are found in hepatopancreatic tissues (Fig. 3B). T2 and T3 show more marked improvement of hepatopancreatic tissues (Fig. 3C and D). The intestinal tilapia transverse sections are represented in Figs. 4 and 5. The distortion mucosa layer of intestine fish is shown in Fig. 4A. Moderate distortion of apical parts of intestinal villi and absorption zone (Fig. 4B). Markedly improved branched and apical parts of intestinal mucosa in T2 and T3 groups (Fig. 4C and D) according to the nano-selenium levels in the experimental diet (Fig. 5) represented the different significant P < 0.05 that showing the mean of villus (mucosal fold) length is improved as 135.6 μm ± 2.561, 136.8 μm ± 3.917 and 137.4 μm ± 3.295 for T1, T2 and T3 respectively that compared to T0 (control) is 115.2 μm ± 4.684. The mean of intestinal villus width is increased to 42.2 μm ± 3.499, 45.4 μm ± 2.619 and 46.2 μm ± 2.131 for T1, T2 and T3 respectively that compared to T0 (control) is 29.6 μm ± 1.806. The mean intestinal muscular fibres layer thickness is upregulation as 32.8 μm ± 1.655, 35 μm ± 2.121 and 36.4 μm ± 2.676 for T1, T2 and T3 respectively that compared to T0 (control) is 28.6 μm ± 1.208. The mean intestinal absorption zone is ameliorative as 7110.8 μm2 ± 363.94, 7615.4 μm2 ± 364.754 and 8212.2 μm2 ± 488.07 for T1, T2 and T3 respectively that compared to T0 (control) is 4787.4 μm2 ± 395.08.

Fig. 3
figure 3

Liver histological sections of Nile tilapia (O. niloticus) fed with each experimental diet. A control (T0), B T1, C T2 and D T3. HC = hepatic cells, S = hepatic sinusoids, P = hepatopancreatic tissue, D = marked degeneration of hepatocytes and arrows = immune cells and mild congestion in the pancreatic islands (H&E, scale bar = 50 μm)

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Fig. 4
figure 4

Intestinal histological sections of Nile tilapia (O. niloticus) fed with the experimental diets. A Control (T0), B T1, C T2 and D T3. S = serosa layer, MF = muscularis fibres layer, SM = submucosal layer, M = mucosal layer, V = intestinal villi and arrows = distortion of some apical parts of intestinal villi (H&E, scale bar = 50 μm)

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Fig. 5
figure 5

Column charts representing the intestinal morphology as villus (mucosal fold) length, villus width, muscular fibres layer thickness and intestinal absorption zone for Nile tilapia O. niloticus after being challenged with Aspergillus flavus infection in different experimental groups. Means within the same column with different superscript letters (a–b) are highly significant at villus length, villus width and intestinal absorption zone P ≤ 0.001 and non-significant at muscular fibres layer thickness P > 0.05 compared between experimental groups (mean ± SE, 𝑁 = 5)

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Transverse sections of fish gills in Fig. 6A and B show demolition of the gill architectures. Figure 6C and D show some mild remediation of primary and secondary gill lamellae and some laceration of gill arches. Figure 6E and F shows some improvements of primary and secondary gill lamellae and gill arches. Figure 6G and H show markedly ameliorated of the gills architecture with some laceration of gill arches and improvement of fish gills. Figure 6H appears mucoid epidermis with some degenerated, necrosis of the muscle layer and edema with leukocytic cells’ infiltration. The sections of skin and skeletal muscles for Nile tilapia are described in Figs. 7 and 8. The highly significant P > 0.001 that showing the mean of villus length is improved as 8 μm ± 0.70, 6.8 μm ± 0.37 and 6.6 μm ± 0.51 for T1, T2 and T3 respectively that compared to T0 (control) is 12.6 μm ± 0.92. Vertical sections in fish skin and skeletal muscles in Fig. 7A show destruction of the skin structure (control group). Figure 7B shows some remodelling of the skin laceration. Figure 7C shows a decrease in the spacing between skin and skeletal muscle fibres. Figure 7D more ameliorated in the skin layers and least spacing between the skin and skeletal muscle fibres.

Fig. 6
figure 6

Histological sections of Nile tilapia (O. niloticus) gills which fed with the experimental diets. A Control (T0), B T1, C T2 and D T3. Gill filament, gill lamellae and asterisks = gill arches (H&E, scale bar = 20 μm)

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Fig. 7
figure 7

Histological sections of Nile tilapia (O. niloticus) skin that fed with the experimental diets. A Control (T0), B T1, C T2 and D T3. D = dermis, S = spacing between layers of skin and muscles (H&E, scale bar = 50 μm)

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Fig. 8
figure 8

Column charts representing the spacing between skin and skeletal muscles for Nile tilapia O. niloticus after being challenged with Aspergillus flavus infection in the different experimental groups. Means within the same column with different superscript letters (a–b) are significantly different at P < 0.001 compared between experimental groups (mean ± SE, 𝑁 = 5)

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Discussion

Selenium nanoparticles are characterized and determined by studying their shape and sizes (Durigon et al. 2019). The small size and vast surface of selenium nanoparticles result in high availability and permeability in fish bodies (Hasimuna et al. 2020). The size distribution of the spherical Se-NPs ranges from 8.28 to 58.88 nm (El-Ouny et al. 2023). This makes them be easily absorbed in the fish body tissues.

Growth indices and fish survival

Supplementation of selenium nanoparticles enhanced growth rate in O. niloticus (Adam et al. 2023), Carassius auratus gibelio (Zhou et al. 2009), Oncorhynchus mykiss (Hunt et al. 2011) and Dicentrarchus labrax (Betancor et al. 2012). Likewise, Ibrahim et al. (Ibrahim et al. 2021) found that the inclusion either bulk selenium (Bulk-Se) or nano-selenium (nano-Se) improved growth performance, feed utilization efficiency and biological parameters of Nile tilapia. The improvement of growth performance and feed conversion ratio of the present study could be attributed to different scenario as (i) nano-Se enhanced gastrointestinal tract morphology and histology, then, improved the metabolism and nutrient assimilation, (ii) Se plays a major role as functional cofactors for endogenous digestive enzymes synthesis (Mansour et al. 2017) which consequently improve nutrient digestion and enhanced growth performance and feed efficiency, (iii) selenium enhances protein digestibility and utilization through boosting the quantity and activity of intestinal microbes and digestive protease activity (Dawood et al. 2019), (iv) adding nano-Se to fish diets heightened the mucosal length/width the number of goblet cells in the intestinal epithelium of fish as well as increased cell proliferation and protein synthesis. This consequently improves the surface absorption area of alimentary canal (Ibrahim et al. 2021), (v) Se performs a crucial function as a co-enzyme’s components for the activation of the gut microbiome in the intestine as well as intestinal enzymes, which improves topography of the gut and capacity of absorption, consequently improve performance and feed efficiency of animals (Mansour et al. 2017) and (vi) Se increases the amount of protein in cells of digestive track which stimulates the absorption and assimilation of nutrient (Dawood et al. 2020).

On the contrary to the present findings, Durigon et al. (Durigon et al. 2019) reported no improvement in growth parameters (weight, TL, SL, SGR and TWG) between the different treatments and the control diet. The differences from previous and current results could be attributed to several factors as trial conditions, fish species and differences in chemical forms used as ordinary selenium is not bioavailable as nanoselenium particles.

Water quality parameters

The cornerstone to an aquaculture business is water quality, and it is commonly claimed that before farming an aquatic life, you should first farm the water (Hasimuna et al. 2020). El-Ouny et al. (El-Ouny et al.,2023) reported that physicochemical parameters of fish pond water are crucial indicators of water quality and a culture system’s capacity to maintain the cultivated species. In the present study, there were no variations in all the critical water quality factors such as temperature, DO, NH4, NO2, NO3, pH and salinity. All estimated water quality variables were within the normal ranges for tilapia culture as indicated in previous studies conducted by (El-Ouny et al. 2023; Mechlaoui et al. 2019). The present results are consistent with Zhou et al. (Zhou et al. 2009), who reported that there was no result of different selenium nanoparticle levels on water quality parameters.

Proximate composition of the fish body

The results of this study demonstrated that body chemical compositions and selenium residues in tissues of tilapia fingerlings were affected (P ≤ 0.05) by nano-Se supplementation. These observations are consistent with Zhou et al. (Zhou et al. 2009) and Le and Fotedar (Rathore et al. 2021) for crucian carp and juvenile yellowtail kingfish, respectively. Furthermore, Ibrahim et al. (Ellis 1990) found that chemical composition of fish and flesh quality was considerably altered by the dietary Bulk-Se or Nano-Se, implying that the tilapia flesh composition is susceptible to addition dietary Se. Likewise, the muscle Se concentrations in carp fed 2 mg nano-Se were higher than the control diet and other treatments (Ashouri et al. 2015). In this concern, the inclusion of organic Se increases the deposition of Se in the muscles of rainbow trout (Oncorhynchus mykiss) compared with the inorganic type (Rider et al. 2009).

Haemato-biochemical parameters

The haemato-biochemical parameters are crucial in determining the health status of aquatic animals (Adam et al. 2023). The current investigation showed that the lymphocytes and neutrophils significantly increased (P ≤ 0.05) in the control more than in other treatments. The present findings are in parallel with Rathore et al. (Rathore et al. 2021), who reported the function of Selenium to modify and regulate and expression of immune-regulated selenoprotein in O. niloticus. In addition, Dawood et al. (Dawood et al. 2019) explained that European seabass have strongly cell-mediated immunity stimulated by supplementation with Nano-Se.

Contrary to the present findings, Ibrahim et al. (Ibrahim et al. 2021) reported no major (P ≥ 0.05) differences in biological indices as well as the values of MCH, MCHC, MCV, MPV and OCCB among experimental diets. In addition, the present study showed that the globulin was considerably augmented by the inclusion of nano-Se. This improvement may be due to the role of selenium to increase the high protein level in serum levels in the intracellular intestines of tilapia. The present results are consistent with Ibrahim et al. (Ibrahim et al. 2021) in Nile tilapia who reported that inclusion of nano-selenium increases serum globulin content. Accordingly, diets fortified with nano-Se had positive effects on the health of fish under culture conditions.

Digestive enzyme activities

In the current work, the highest activity of lipase was found in diet enriched with Se compared to the control diets, while amylase and protease activities were not significantly affected with the inclusion of selenium. The present results are consistent with Adineh et al. (Adineh et al. 2021), who noted that the inclusion of nano-Se up to (2 mg kg−1) can substantially enhance digestive enzyme in grass carp. Furthermore, Ibrahim et al. (Ibrahim et al. 2021) found that the inclusion of either nano or bulk selenium in Nile tilapia diets improved digestive enzyme activity. The role of selenium in improving and enhancing digestive enzymes activity could be attributed to Se function as co-enzymes in the synthesis/activation of digestive enzymes (Mansour et al. 2017).

Antioxidants and immunity biomarkers

Selenium nanoparticles are known to act as a foundation for antioxidative enzyme generation leading to high total antioxidative potency. The present work displayed improvement in the activities of MDA, GPX, CAT and SOD. Lysozyme and IgM of fish fed diet augmented with nano-Se than the control group. The current findings are consistent with previous analyses that described supplementation of nano-Se developed antioxidant parameters for common carp (Ashouri et al. 2015) and Nile tilapia (Dawood et al. 2020). Furthermore, European sea bass (Dicentrarchus labrax) fed diet supplemented with selenium nanoparticle decrease the oxidative stress and improve oxidative enzymes activity (Betancor et al. 2012). In addition, inclusion of selenium nanoparticle improved cellular shield against oxidative stress and an increase in antioxidant defences of fish when exposed to disease-causing pathogens (Durigon et al. 2019). Furthermore, Sarkar et al. (Durigon et al. 2019) reported that nano-Se can be fed to fish and livestock to decrease oxidative stress as well as to increase the productivity of the stressed fish and livestock. Likewise, Ibrahim et al. (Ibrahim et al. 2021) found that Nile tilapia fed 0.4 or 0.8 mg nano-Se improved the performance of antioxidant enzymes and lower MDA activity.

Challenge test with Aspergillus flavus

The challenge test of Oreochromis niloticus with Aspergillus flavus revealed a decreasing trend of mortality rates (i.e. 70%, 60%, 40.33% and 40.67% in control, T1, T2 and T3 groups respectively). The present results are consistent Pérez-Valenzuela et al. (Pérez-Valenzuela et al. 2021) who found that Oncorhynchus mykiss fed diets enriched with selenium lowered the cytopathic effect, mortality tare, intracellular bacterial load and cellular mortality of SHK-1 by expanding the abundance and activity of host glutathione peroxidase. Eissa et al. (Eissa et al. 2022b; Eissa et al. 2023b) found an increase in resistance to Aspergillus flavus of Oreochromis niloticus and Red Tilapia (Oreochromis sp.) fed diets supplemented with curcumin nanoparticles due to an increase of antioxidant capacity in fish’s plasma.

Histology findings

The present results are consistent with Mello et al. (Mello et al. 2013), who found that Se-NP addition elevated the quantity and activity of gut microbes and digestive protease potency. Furthermore, the insertion of nano-Se in diets enlarged the villus length/width and the number of goblet cells in intestinal fish as well as amplified cell proliferation and protein synthesis, which consequently improve the surface absorption area of the alimentary canal; Se plays a crucial role as a co-enzyme factor for the activation of the digestive enzymes and the gut flora, which amended topography of gut and capacity of absorption, which subsequently improve performance and feed efficiency of animals. Nano-selenium may increase the content of protein in cells of the intestinal tract, which expands the absorption and assimilation of nutrients. Nano-selenium was able to influence tilapia infection against Aspergillus flavus. The T3 group markedly improved the hepatopancreatic tissue, intestinal layers, gill tissues and fish skin condition. This most likely increased the immune resistance of Nile tilapia and Red Tilapia challenged with Aspergillus flavus infection. This corresponds to the findings in recent studies with fish (Eissa et al. 2022b; Eissa et al. 2023b). Finally, this work has highlighted the potential of nano-mineral delivery in terms of achieving superior Se uptake and assimilation in tilapia. This complies with the recent benefits in aquaculture, as recently reviewed by Nasr-Eldahan et al. (Nasr-Eldahan et al. 2021), to enhance disease resistance and health in farmed fish species.

Conclusion

The supplementation of selenium nanoparticles in diets of O. niloticus significantly improved the fish’s growth performance survival rate, boost immune system response, antioxidant capacity and digestive enzymes with selenium nanoparticles up to 1.0 mg/kg diet that linked to reduce the mortality rate when the fish was challenged with A. flavus.

Recommendation

This is crucial study which recommends more studies be undertaken to investigate the effect of a combination of selenium nanoparticles and vitamin premixes on growth performance, enzymatic activities and immune system response. In addition, more research in the future is recommended to clarify the function of selenium nanoparticles on water quality parameters that could help to achieve aquaculture sustainability of fish farms from different regions (temperate and tropical) should expect in their fish production businesses.